A pressing need exists for clinically applicable systems for ex vivo platelet production. Multiple technological advances have set the stage to attain this goal. Inducible pluripotent stem cells (iPSC) derived from donor somatic cells can now be manipulated to yield customized, expandable megakaryocyte (Mk) progenitors. Perfusion bioreactors that replicate the bone marrow environment have enhanced the efficiency of functional platelet release and collection. The biggest roadblock in clinical translation consiss of the problem of scalability. In particular, highly proliferative Mk progenitors yield poor platelt numbers, and Mk with high platelet yields come from progenitors with very limited prolferative capacity. An ability to circumvent these limitations by combining progenitor expandability with efficient platelet production will be critical for cost- effective scale-up. The efficiency of platlet production depends on a program of Mk morphogenesis involving massive cellular enlargement and polyploidization. The relative balance of morphogenetic versus proliferative potential depends on ontogenic developmental stage. Thus, fetal and neonatal Mk progenitors show extensive self-renewal but limited morphogenesis. Progenitors derived from human ESC and iPSC recapitulate the features of early fetal megakaryopoiesis: high proliferation with minimal morphogenesis. The influence of ontogenic stage affects not only platelet numbers but also extends to platelet function. Specifically, Mk from earlier in ontogeny yield platelets with proportionally diminished aggregation capacity. Our lab identified a signaling pathway critical in Mk morphogenesis (Elagib et al. Dev. Cell, 2013). In this pathway, high-amplitude activation of the transcriptional kinase P-TEFb occurs due to downregulation of the noncoding RNA 7SK. In new unpublished data, we find that defects in this Mk morphogenesis pathway underlie the phenotypic differences between neonatal and adult Mk. Specifically, neonatal Mk fail to downregulate 7SK and fail to trigger high-amplitude activation of P-TEFb. We have identified a 7SK binding factor, IGF2BP3, that is present only in neonatal Mk and functions as an ontogenic masterswitch in Mk morphogenesis. Antagonism of IGF2BP3 by either shRNA knockdown or a novel inhibitory compound significantly augments morphogenesis in neonatal Mk. Conversely, ectopic IGF2BP3 converts adult MK into a fetal phenotype, and a putative agonist compound augments fetal-like features in neonatal Mk. IGF2BP3 thus represents a highly attractive target for engineering scalable megakaryopoiesis. In human neonatal Mk, it appears to be the key determinant of Mk ontogenic phenotype. From a therapeutic perspective, it is a druggable target, with the capability of both negative and positive modulation. Aim 1 will examine the morphogenesis signaling pathway in iPSC Mk, the contributions of IGF2BP factors, and optimal approaches to enhance thrombopoiesis. Aim 2 will take a complementary approach and determine circuits necessary for fetal reprogramming of adult Mk progenitors, thereby allowing for their large-scale, reversible expansion.